RESUMO
Thousands of cis-elements in genomes are predicted to have vital functions. Although conservation, activity in surrogate assays, polymorphisms, and disease mutations provide functional clues, deletion from endogenous loci constitutes the gold-standard test. A GATA-2-binding, Gata2 intronic cis-element (+9.5) required for hematopoietic stem cell genesis in mice is mutated in a human immunodeficiency syndrome. Because +9.5 is the only cis-element known to mediate stem cell genesis, we devised a strategy to identify functionally comparable enhancers ("+9.5-like") genome-wide. Gene editing revealed +9.5-like activity to mediate GATA-2 occupancy, chromatin opening, and transcriptional activation. A +9.5-like element resided in Samd14, which encodes a protein of unknown function. Samd14 increased hematopoietic progenitor levels/activity and promoted signaling by a pathway vital for hematopoietic stem/progenitor cell regulation (stem cell factor/c-Kit), and c-Kit rescued Samd14 loss-of-function phenotypes. Thus, the hematopoietic stem/progenitor cell cistrome revealed a mediator of a signaling pathway that has broad importance for stem/progenitor cell biology.
Assuntos
Fator de Transcrição GATA2/genética , Células-Tronco Hematopoéticas/metabolismo , Proteínas/genética , Proteínas Proto-Oncogênicas c-kit/genética , Ativação Transcricional/genética , Sequência de Aminoácidos , Animais , Diferenciação Celular/genética , Linhagem Celular , Camundongos , Dados de Sequência Molecular , Proteínas/metabolismo , Interferência de RNA , RNA Interferente Pequeno , Transdução de Sinais , Transcrição Gênica/genéticaRESUMO
RNA-seq is currently the technology of choice for global measurement of transcript abundances in cells. Despite its successes, isoform-level quantification remains difficult because short RNA-seq reads are often compatible with multiple alternatively spliced isoforms. Existing methods rely heavily on uniquely mapping reads, which are not available for numerous isoforms that lack regions of unique sequence. To improve quantification accuracy in such difficult cases, we developed a novel computational method, prior-enhanced RSEM (pRSEM), which uses a complementary data type in addition to RNA-seq data. We found that ChIP-seq data of RNA polymerase II and histone modifications were particularly informative in this approach. In qRT-PCR validations, pRSEM was shown to be superior than competing methods in estimating relative isoform abundances within or across conditions. Data-driven simulations suggested that pRSEM has a greatly decreased false-positive rate at the expense of a small increase in false-negative rate. In aggregate, our study demonstrates that pRSEM transforms existing capacity to precisely estimate transcript abundances, especially at the isoform level.
Assuntos
Processamento Alternativo/genética , RNA/genética , Análise de Sequência de RNA/métodos , Algoritmos , Biologia Computacional/métodos , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , RNA Polimerase II/genética , SoftwareRESUMO
The unremitting demand to replenish differentiated cells in tissues requires efficient mechanisms to generate and regulate stem and progenitor cells. Although master regulatory transcription factors, including GATA binding protein-2 (GATA-2), have crucial roles in these mechanisms, how such factors are controlled in developmentally dynamic systems is poorly understood. Previously, we described five dispersed Gata2 locus sequences, termed the -77, -3.9, -2.8, -1.8, and +9.5 GATA switch sites, which contain evolutionarily conserved GATA motifs occupied by GATA-2 and GATA-1 in hematopoietic precursors and erythroid cells, respectively. Despite common attributes of transcriptional enhancers, targeted deletions of the -2.8, -1.8, and +9.5 sites revealed distinct and unpredictable contributions to Gata2 expression and hematopoiesis. Herein, we describe the targeted deletion of the -3.9 site and mechanistically compare the -3.9 site with other GATA switch sites. The -3.9(-/-) mice were viable and exhibited normal Gata2 expression and steady-state hematopoiesis in the embryo and adult. We established a Gata2 repression/reactivation assay, which revealed unique +9.5 site activity to mediate GATA factor-dependent chromatin structural transitions. Loss-of-function analyses provided evidence for a mechanism in which a mediator of long-range transcriptional control [LIM domain binding 1 (LDB1)] and a chromatin remodeler [Brahma related gene 1 (BRG1)] synergize through the +9.5 site, conferring expression of GATA-2, which is known to promote the genesis and survival of hematopoietic stem cells.
Assuntos
Fator de Transcrição GATA2/metabolismo , Sequências Reguladoras de Ácido Nucleico , Células-Tronco/citologia , Animais , Sequência de Bases , Diferenciação Celular/genética , Células Cultivadas , Elementos Facilitadores Genéticos , Hematopoese , Humanos , Íntrons , Camundongos , Dados de Sequência Molecular , Homologia de Sequência do Ácido Nucleico , Células-Tronco/metabolismoRESUMO
Previous reports of GATA2 mutations have focused on the coding region of the gene or full gene deletions. We recently identified 2 patients with novel insertion/deletion mutations predicted to result in mRNA nonsense-mediated decay, suggesting haploinsufficiency as the mechanism of GATA2 deficient disease. We therefore screened patients without identified exonic lesions for mutations within conserved noncoding and intronic regions. We discovered 1 patient with an intronic deletion mutation, 4 patients with point mutations within a conserved intronic element, and 3 patients with reduced or absent transcription from 1 allele. All mutations affected GATA2 transcription. Full-length cDNA analysis provided evidence for decreased expression of the mutant alleles. The intronic deletion and point mutations considerably reduced the enhancer activity of the intron 5 enhancer. Analysis of 512 immune system genes revealed similar expression profiles in all clinically affected patients and reduced GATA2 transcript levels. These mutations strongly support the haploinsufficient nature of GATA2 deficiency and identify transcriptional mechanisms and targets that lead to MonoMAC syndrome.
Assuntos
Fator de Transcrição GATA2/genética , Haploinsuficiência/genética , Leucopenia/genética , Mutação/fisiologia , Infecção por Mycobacterium avium-intracellulare/genética , Adolescente , Adulto , Idoso , Sequência de Bases , Criança , Pré-Escolar , Sequência Conservada/genética , Feminino , Humanos , Lactente , Íntrons/genética , Células K562 , Leucopenia/sangue , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Monócitos/patologia , Infecção por Mycobacterium avium-intracellulare/sangue , Degradação do RNAm Mediada por Códon sem Sentido/genética , Síndrome , Adulto JovemRESUMO
PURPOSE OF REVIEW: Erythropoiesis, in which hematopoietic stem cells (HSCs) generate lineage-committed progenitors that mature into erythrocytes, is regulated by numerous chromatin modifying and remodeling proteins. We will focus on how epigenetic and genetic mechanisms mesh to establish the erythroid transcriptome and how studying erythropoiesis can yield genomic principles. RECENT FINDINGS: Trans-acting factor binding to small DNA motifs (cis-elements) underlies regulatory complex assembly at specific chromatin sites, and therefore unique transcriptomes. As cis-elements are often very small, thousands or millions of copies of a given element reside in a genome. Chromatin restricts factor access in a context-dependent manner, and cis-element-binding factors recruit chromatin regulators that mediate functional outputs. Technologies to map chromatin attributes of loci in vivo, to edit genomes and to sequence whole genomes have been transformative in discovering critical cis-elements linked to human disease. SUMMARY: Cis-elements mediate chromatin-targeting specificity, and chromatin regulators dictate cis-element accessibility/function, illustrating an amalgamation of genetic and epigenetic mechanisms. Cis-elements often function ectopically when studied outside of their endogenous loci, and complex strategies to identify nonredundant cis-elements require further development. Facile genome-editing technologies provide a new approach to address this problem. Extending genetic analyses beyond exons and promoters will yield a rich pipeline of cis-element alterations with importance for red cell biology and disease.
Assuntos
Epigenômica , Eritrócitos/fisiologia , Fatores de Transcrição/fisiologia , Perfilação da Expressão Gênica , Hematopoese/fisiologia , HumanosRESUMO
CIC-DUX4-rearranged sarcoma (CDS) is a rare and aggressive soft tissue tumor that occurs most frequently in young adults. The key oncogenic driver of this disease is the expression of the CIC-DUX4 fusion protein as a result of chromosomal rearrangements. CIC-DUX4 displays chromatin binding properties, and is therefore believed to function as an aberrant transcription factor. However, the chromatin remodeling events induced by CIC-DUX4 are not well understood, limiting our ability to identify new mechanism-based therapeutic strategies for these patients. Here, we generated a genome-wide profile of CIC-DUX4 DNA occupancy and associated chromatin states in human CDS cell models and primary tumors. Combining chromatin profiling, proximity ligation assays, as well as genetic and pharmacological perturbations, we show that CIC-DUX4 operates as a potent transcriptional activator at its binding sites. This property is in contrast with the repressive function of the wild-type CIC protein, and is mainly mediated through the direct interaction of CIC-DUX4 with the acetyltransferase p300. In keeping with this, we show p300 to be essential for CDS tumor cell proliferation; additionally, we find its pharmacological inhibition to significantly impact tumor growth in vitro and in vivo. Taken together, our study elucidates the mechanisms underpinning CIC-DUX4-mediated transcriptional regulation.
RESUMO
The development of targeted therapies offers new hope for patients affected by incurable cancer. However, multiple challenges persist, notably in controlling tumor cell plasticity in patients with refractory and metastatic illness. Neuroblastoma (NB) is an aggressive pediatric malignancy originating from defective differentiation of neural crest-derived progenitors with oncogenic activity due to genetic and epigenetic alterations and remains a clinical challenge for high-risk patients. To identify critical genes driving NB aggressiveness, we performed combined chromatin and transcriptome analyses on matched patient-derived xenografts (PDXs), spheroids, and differentiated adherent cultures derived from metastatic MYCN nonamplified tumors. Bone marrow kinase on chromosome X (BMX) was identified among the most differentially regulated genes in PDXs and spheroids versus adherent models. BMX expression correlated with high tumor stage and poor patient survival and was crucial to the maintenance of the self-renewal and tumorigenic potential of NB spheroids. Moreover, BMX expression positively correlated with the mesenchymal NB cell phenotype, previously associated with increased chemoresistance. Finally, BMX inhibitors readily reversed this cellular state, increased the sensitivity of NB spheroids toward chemotherapy, and partially reduced tumor growth in a preclinical NB model. Altogether, our study identifies BMX as a promising innovative therapeutic target for patients with high-risk MYCN nonamplified NB.
Assuntos
Proteína Proto-Oncogênica N-Myc , Neuroblastoma , Esferoides Celulares , Neuroblastoma/genética , Neuroblastoma/patologia , Neuroblastoma/tratamento farmacológico , Neuroblastoma/metabolismo , Humanos , Proteína Proto-Oncogênica N-Myc/genética , Proteína Proto-Oncogênica N-Myc/metabolismo , Animais , Esferoides Celulares/patologia , Esferoides Celulares/metabolismo , Esferoides Celulares/efeitos dos fármacos , Camundongos , Ensaios Antitumorais Modelo de Xenoenxerto , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão GênicaRESUMO
Tlx3 (HOX11L2) is regarded as one of the selector genes in excitatory versus inhibitory fate specification of neurons in distinct regions of the nervous system. Expression of Tlx3 in a post-mitotic immature neuron favors a glutamatergic over GABAergic fate. The factors that regulate Tlx3 have immense importance in the fate specification of glutamatergic neurons. Here, we have shown that Notch target gene, Hes-1, negatively regulates Tlx3 expression, resulting in decreased generation of glutamatergic neurons. Down-regulation of Hes-1 removed the inhibition on Tlx3 promoter, thus promoting glutamatergic differentiation. Promoter-protein interaction studies with truncated/mutated Hes-1 protein suggested that the co-repressor recruitment mediated through WRPW domain of Hes-1 has contributed to the repressive effect. Our results clearly demonstrate a new and unique role for canonical Notch signaling through Hes-1, in neurotransmitter/subtype fate specification of neurons in addition to its known functional role in proliferation/maintenance of neural progenitors.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Regulação da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Células-Tronco Neurais/citologia , Sequência de Aminoácidos , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Sítios de Ligação , Diferenciação Celular , Linhagem Celular , DNA/metabolismo , Células-Tronco Embrionárias/citologia , Ácido Glutâmico/farmacologia , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/genética , Humanos , Camundongos , Dados de Sequência Molecular , Células-Tronco Neurais/metabolismo , Neurônios/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Estrutura Terciária de Proteína , Transdução de Sinais , Fatores de Transcrição HES-1 , Ácido gama-Aminobutírico/farmacologiaRESUMO
Developmental control mechanisms often use multimeric complexes containing transcription factors, coregulators, and additional non-DNA binding components. It is challenging to ascertain how such components contribute to complex function at endogenous loci. We analyzed the function of components of a complex containing master regulators of hematopoiesis (GATA-1 and Scl/TAL1) and the non-DNA binding components ETO2, the LIM domain protein LMO2, and the chromatin looping factor LDB1. Surprisingly, we discovered that ETO2 and LMO2 regulate distinct target-gene ensembles in erythroid cells. ETO2 commonly repressed GATA-1 function via suppressing histone H3 acetylation, although it also regulated methylation of histone H3 at lysine 27 at select loci. Prior studies defined multiple modes by which GATA-1 regulates target genes with or without the coregulator Friend of GATA-1 (FOG-1). LMO2 selectively repressed genes that GATA-1 represses in a FOG-1-independent manner. As LMO2 controls hematopoiesis, its dysregulation is leukemogenic, and its influence on GATA factor function is unknown, this mechanistic link has important biological and pathophysiological implications. The demonstration that ETO2 and LMO2 exert qualitatively distinct functions at endogenous loci illustrates how components of complexes containing master developmental regulators can impart the capacity to regulate unique cohorts of target genes, thereby diversifying complex function.
Assuntos
Hematopoese/genética , Complexos Multiproteicos/fisiologia , Elementos Reguladores de Transcrição/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Western Blotting , Células Cultivadas , Imunoprecipitação da Cromatina , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fator de Transcrição GATA1/genética , Fator de Transcrição GATA1/metabolismo , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Proteínas com Domínio LIM , Metaloproteínas/genética , Metaloproteínas/metabolismo , Complexos Multiproteicos/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , RNA Interferente Pequeno/genética , Proteína 1 Parceira de Translocação de RUNX1 , Elementos Reguladores de Transcrição/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína 1 de Leucemia Linfocítica Aguda de Células T , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Cell fate transitions observed in embryonic development involve changes in three-dimensional genomic organization that provide proper lineage specification. Whether similar events occur within tumor cells and contribute to cancer evolution remains largely unexplored. We modeled this process in the pediatric cancer Ewing sarcoma and investigated high-resolution looping and large-scale nuclear conformation changes associated with the oncogenic fusion protein EWS-FLI1. We show that chromatin interactions in tumor cells are dominated by highly connected looping hubs centered on EWS-FLI1 binding sites, which directly control the activity of linked enhancers and promoters to establish oncogenic expression programs. Conversely, EWS-FLI1 depletion led to the disassembly of these looping networks and a widespread nuclear reorganization through the establishment of new looping patterns and large-scale compartment configuration matching those observed in mesenchymal stem cells, a candidate Ewing sarcoma progenitor. Our data demonstrate that major architectural features of nuclear organization in cancer cells can depend on single oncogenes and are readily reversed to reestablish latent differentiation programs.
Assuntos
Sarcoma de Ewing , Criança , Humanos , Sarcoma de Ewing/genética , Sarcoma de Ewing/metabolismo , Sarcoma de Ewing/patologia , Cromatina/genética , Linhagem Celular Tumoral , Proteína EWS de Ligação a RNA/genética , Proteína EWS de Ligação a RNA/metabolismo , Sítios de Ligação , Diferenciação Celular , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Regulação Neoplásica da Expressão GênicaRESUMO
Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) is rapidly replacing chromatin immunoprecipitation combined with genome-wide tiling array analysis (ChIP-chip) as the preferred approach for mapping transcription-factor binding sites and chromatin modifications. The state of the art for analyzing ChIP-seq data relies on using only reads that map uniquely to a relevant reference genome (uni-reads). This can lead to the omission of up to 30% of alignable reads. We describe a general approach for utilizing reads that map to multiple locations on the reference genome (multi-reads). Our approach is based on allocating multi-reads as fractional counts using a weighted alignment scheme. Using human STAT1 and mouse GATA1 ChIP-seq datasets, we illustrate that incorporation of multi-reads significantly increases sequencing depths, leads to detection of novel peaks that are not otherwise identifiable with uni-reads, and improves detection of peaks in mappable regions. We investigate various genome-wide characteristics of peaks detected only by utilization of multi-reads via computational experiments. Overall, peaks from multi-read analysis have similar characteristics to peaks that are identified by uni-reads except that the majority of them reside in segmental duplications. We further validate a number of GATA1 multi-read only peaks by independent quantitative real-time ChIP analysis and identify novel target genes of GATA1. These computational and experimental results establish that multi-reads can be of critical importance for studying transcription factor binding in highly repetitive regions of genomes with ChIP-seq experiments.
Assuntos
Imunoprecipitação da Cromatina/métodos , Genômica/métodos , Sequências Repetitivas de Ácido Nucleico , Análise de Sequência de DNA/métodos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Animais , Composição de Bases , Sítios de Ligação , Mapeamento Cromossômico/métodos , Fator de Transcrição GATA1/genética , Genoma , Células HeLa , Ensaios de Triagem em Larga Escala/métodos , Humanos , Camundongos , Reprodutibilidade dos Testes , Fator de Transcrição STAT1/genéticaRESUMO
Evolutionarily conserved Notch signaling orchestrates diverse physiological mechanisms during metazoan development and homeostasis. Classically, ligand-activated Notch receptors transduce the signaling cascade through the interaction of DNA-bound CBF1-co-repressor complex. However, recent reports have demonstrated execution of a CBF1-independent Notch pathway through signaling cross-talks in various cells/tissues. Here, we have tried to congregate the reports that describe the non-canonical/CBF1-independent Notch signaling and target gene activation in vertebrates with specific emphasis on their functional relevance.
Assuntos
Regulação da Expressão Gênica/fisiologia , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais/fisiologia , Vertebrados/metabolismo , Animais , Humanos , Modelos BiológicosRESUMO
Hes-1 and Hes-5 are downstream effectors of Notch signaling that are known to be involved in different aspects of neural stem cell proliferation and differentiation. Evidence has emerged that Hes-1 expression can be regulated by alternate signaling pathways independent of canonical Notch/CBF1 interaction. This context-dependent differential regulation of Hes-1 expression in neural progenitor gains a lot of importance as it would help in its exponential expansion without the requirement of interaction from neighboring cells during development. Here, we have clearly demonstrated the existence of a population of neural progenitors with Notch/CBF1-independent Hes-1 expression in vitro. Further analysis demonstrated the role of FGF2 in activating Hes-1 expression through the direct binding of ATF2, a JNK downstream target, on Hes-1 promoter. This raises the possibility for the existence of two distinct populations of neural progenitors - one maintained by Hes-1 expression exclusively through Notch-independent mechanism and the other mediating Hes-1 expression through both canonical Notch and FGF2-ATF2 pathway. This alternative pathway will insure a constant expression of Hes-1 even in the absence of canonical Notch intracellular domain-mediated signaling, thereby maintaining a pool of proliferating neural progenitors required during development.
Assuntos
Fator 2 Ativador da Transcrição/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Sistema Nervoso Central/embriologia , Proteínas de Homeodomínio/metabolismo , Neurônios/metabolismo , Receptores Notch/metabolismo , Células-Tronco/metabolismo , Fator 2 Ativador da Transcrição/genética , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Diferenciação Celular/fisiologia , Linhagem Celular , Fator 2 de Crescimento de Fibroblastos/genética , Fator 2 de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas de Homeodomínio/genética , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/genética , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Camundongos , Neurônios/citologia , Regiões Promotoras Genéticas/genética , Receptores Notch/genética , Transdução de Sinais/fisiologia , Células-Tronco/citologia , Fatores de Transcrição HES-1 , Ativação Transcricional/fisiologiaRESUMO
Adult hippocampal neurogenesis is altered in response to different physiological and pathological stimuli. GFAP(+ve)/nestin(+ve) radial glial like Type-1 progenitors are considered to be the resident stem cell population in adult hippocampus. During neurogenesis these Type-1 progenitors matures to GFAP(-ve)/nestin(+ve) Type-2 progenitors and then to Type-3 neuroblasts and finally differentiates into granule cell neurons. In our study, using pilocarpine-induced seizure model, we showed that seizure initiated activation of multiple progenitors in the entire hippocampal area such as DG, CA1 and CA3. Seizure induction resulted in activation of two subtypes of Type-1 progenitors, Type-1a (GFAP(+ve)/nestin(+ve)/BrdU(+ve)) and Type-1b (GFAP(+ve)/nestin(+ve)/BrdU(-ve)). We showed that majority of Type-1b progenitors were undergoing only a transition from a state of dormancy to activated form immediately after seizures rather than proliferating, whereas Type-1a showed maximum proliferation by 3 days post-seizure induction. Type-2 (GFAP(-ve)/nestin(+ve)/BrdU(+ve)) progenitors were few compared to Type-1. Type-3 (DCX(+ve)) progenitors showed increased expression of immature neurons only in DG region by 3 days after seizure induction indicating maturation of progenitors happens only in microenvironment of DG even though progenitors are activated in CA1 and CA3 regions of hippocampus. Also parallel increase in growth factors expression after seizure induction suggests that microenvironmental niche has a profound effect on stimulation of adult neural progenitors.
Assuntos
Hipocampo/fisiopatologia , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Neurônios/fisiologia , Convulsões/fisiopatologia , Células-Tronco/fisiologia , Animais , Giro Denteado/metabolismo , Giro Denteado/fisiopatologia , Proteínas do Domínio Duplacortina , Proteína Duplacortina , Hipocampo/metabolismo , Masculino , Proteínas Associadas aos Microtúbulos/biossíntese , Neurônios/metabolismo , Neuropeptídeos/biossíntese , Ratos , Ratos Endogâmicos , Convulsões/metabolismo , Células-Tronco/metabolismoRESUMO
Ca(2+) influx through NMDA-type glutamate receptor at excitatory synapses causes activation of post-synaptic Ca(2+)/calmodulin-dependent protein kinase type II (CaMKII) and its translocation to the NR2B subunit of NMDA receptor. The major binding site for CaMKII on NR2B undergoes phosphorylation at Ser1303, in vivo. Even though some regulatory effects of this phosphorylation are known, the mode of dephosphorylation of NR2B-Ser1303 is still unclear. We show that phosphorylation status at Ser1303 enables NR2B to distinguish between the Ca(2+)/calmodulin activated form and the autonomously active Thr286-autophosphorylated form of CaMKII. Green fluorescent protein-alpha-CaMKII co-expressed with NR2B sequence in human embryonic kidney 293 cells was used to study intracellular binding between the two proteins. Binding in vitro was studied by glutathione-S-transferase pull-down assay. Thr286-autophosphorylated alpha-CaMKII or the autophosphorylation mimicking mutant, T286D-alpha-CaMKII, binds NR2B sequence independent of Ca(2+)/calmodulin unlike native wild-type alpha-CaMKII. We show enhancement of this binding by Ca(2+)/calmodulin. Phosphorylation or a phosphorylation mimicking mutation on NR2B (NR2B-S1303D) abolishes the Ca(2+)/calmodulin-independent binding whereas it allows the Ca(2+)/calmodulin-dependent binding of alpha-CaMKII in vitro. Similarly, the autonomously active mutants, T286D-alpha-CaMKII and F293E/N294D-alpha-CaMKII, exhibited Ca(2+)-independent binding to non-phosphorylatable mutant of NR2B under intracellular conditions. We also show for the first time that phosphatases in the brain such as protein phosphatase 1 and protein phosphatase 2A dephosphorylate phospho-Ser1303 on NR2B.
Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Sistema Nervoso Central/metabolismo , Ácido Glutâmico/metabolismo , Neurônios/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Sequência de Aminoácidos/fisiologia , Animais , Sítios de Ligação/fisiologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Linhagem Celular , Proteínas de Fluorescência Verde/genética , Humanos , Insetos , Mutação/genética , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação , Ligação Proteica/fisiologia , Transporte Proteico/fisiologia , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/genética , Proteínas Recombinantes de Fusão/metabolismo , Serina/metabolismo , Transmissão Sináptica/fisiologiaRESUMO
ES cells have been reported to serve as an excellent source for obtaining various specialized cell types and could be used in cell replacement therapy. Here, we demonstrate the potential of ES cells to differentiate along retinal ganglion cell (RGC) lineage. FGF2-induced ES cell derived neural progenitors (ES-NPs) were able to generate RGC-like cells in vitro upon differentiation. These cells expressed RGC regulators and markers such as, Ath5, Brn3b, RPF-1, Thy-1 and Islet-1, confirming their potential to differentiate into RGCs. The generation of RGCs from ES-NPs was enhanced with the exposure of FGF2 and Sonic hedgehog (Shh), although Shh treatment alone did not affect RGC differentiation significantly. ES-NPs, after exposure to FGF2, were capable of integrating and differentiating into RGCs in vivo upon transplantation. Thus, our study suggests that ES cells can serve an excellent renewable source for generating RGCs that can be used to treat neurodegenerative diseases like glaucoma.
Assuntos
Diferenciação Celular , Células-Tronco Embrionárias/fisiologia , Células Ganglionares da Retina/citologia , Animais , Técnicas de Cultura de Células , Linhagem da Célula , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/farmacologia , Glaucoma/cirurgia , Proteínas Hedgehog/farmacologia , Camundongos , Células Ganglionares da Retina/transplanteRESUMO
The master regulatory transcription factor GATA-2 triggers hematopoietic stem and progenitor cell generation. GATA2 haploinsufficiency is implicated in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), and GATA2 overexpression portends a poor prognosis for AML. However, the constituents of the GATA-2-dependent genetic network mediating pathogenesis are unknown. We described a p38-dependent mechanism that phosphorylates GATA-2 and increases GATA-2 target gene activation. We demonstrate that this mechanism establishes a growth-promoting chemokine/cytokine circuit in AML cells. p38/ERK-dependent GATA-2 phosphorylation facilitated positive autoregulation of GATA2 transcription and expression of target genes, including IL1B and CXCL2. IL-1ß and CXCL2 enhanced GATA-2 phosphorylation, which increased GATA-2-mediated transcriptional activation. p38/ERK-GATA-2 stimulated AML cell proliferation via CXCL2 induction. As GATA2 mRNA correlated with IL1B and CXCL2 mRNAs in AML-M5 and high expression of these genes predicted poor prognosis of cytogenetically normal AML, we propose that the circuit is functionally important in specific AML contexts.
Assuntos
Retroalimentação Fisiológica , Fator de Transcrição GATA2/genética , Regulação Leucêmica da Expressão Gênica , Leucemia Mieloide Aguda/genética , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Linhagem Celular Tumoral , Proliferação de Células , Quimiocina CXCL2/genética , Quimiocina CXCL2/metabolismo , Fator de Transcrição GATA2/metabolismo , Haploinsuficiência , Humanos , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Cariotipagem , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/mortalidade , Leucemia Mieloide Aguda/patologia , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação , Prognóstico , Transdução de Sinais , Análise de Sobrevida , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Homeobox gene Tlx3 is known to promote glutamatergic differentiation and is expressed in post-mitotic neurons of CNS. Contrary to this here, we discovered that Tlx3 is expressed in the proliferating progenitors of the external granule layer in the cerebellum, and examined factors that regulate this expression. Using Pax6(-/-)Sey mouse model and molecular interaction studies we demonstrate Pax6 is a key activator of Tlx3 specifically in cerebellum, and induces its expression starting at embryonic day (E)15. By Postnatal day (PN)7, Tlx3 is expressed in a highly restricted manner in the cerebellar granule neurons of the posterior cerebellar lobes, where it is required for the restricted expression of nicotinic cholinergic receptor-α3 subunit (Chrnα3) and other genes involved in formation of synaptic connections and neuronal migration. These results demonstrate a novel role for Tlx3 and indicate that Pax6-Tlx3 expression and interaction is part of a region specific regulatory network in cerebellum and its deregulation during development could possibly lead to Autistic spectral disorders (ASD).
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Células-Tronco Neurais/metabolismo , Neurônios/metabolismo , Fator de Transcrição PAX6/metabolismo , Receptores Nicotínicos/genética , Animais , Diferenciação Celular/genética , Linhagem Celular , Proliferação de Células , Cerebelo/citologia , Cerebelo/metabolismo , Análise por Conglomerados , Imunofluorescência , Perfilação da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Modelos Biológicos , Células-Tronco Neurais/citologia , Neurogênese/genética , Neurônios/citologia , Medula EspinalRESUMO
Erythropoiesis, in which committed progenitor cells generate millions of erythrocytes daily, involves dramatic changes in the chromatin structure and transcriptome of erythroblasts, prior to their enucleation. While the involvement of the master-regulatory transcription factors GATA binding protein 1 (GATA-1) and GATA-2 in this process is established, the mechanistic contributions of many chromatin-modifying/remodeling enzymes in red cell biology remain enigmatic. We demonstrated that SetD8, a histone methyltransferase that catalyzes monomethylation of histone H4 at lysine 20 (H4K20me1), is a context-dependent GATA-1 corepressor in erythroid cells. To determine whether SetD8 controls erythroid maturation and/or function, we used a small hairpin RNA (shRNA)-based loss-of-function strategy in a primary murine erythroblast culture system. In this system, SetD8 promoted erythroblast maturation and survival, and this did not involve upregulation of the established regulator of erythroblast survival Bcl-x(L). SetD8 catalyzed H4K20me1 at a critical Gata2 cis element and restricted occupancy by an enhancer of Gata2 transcription, Scl/TAL1, thereby repressing Gata2 transcription. Elevating GATA-2 levels in erythroid precursors yielded a maturation block comparable to that induced by SetD8 downregulation. As lowering GATA-2 expression in the context of SetD8 knockdown did not rescue erythroid maturation, we propose that SetD8 regulation of erythroid maturation involves multiple target genes. These results establish SetD8 as a determinant of erythroid cell maturation and provide a framework for understanding how a broadly expressed histone-modifying enzyme mediates cell-type-specific GATA factor function.
Assuntos
Epigênese Genética , Células Eritroides/citologia , Fator de Transcrição GATA2/genética , Histona-Lisina N-Metiltransferase/metabolismo , Animais , Sobrevivência Celular , Células Cultivadas , Células Eritroides/metabolismo , Eritropoese , Fator de Transcrição GATA2/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Histona-Lisina N-Metiltransferase/genética , Histonas/metabolismo , Camundongos , Ativação TranscricionalRESUMO
Cis-element encyclopedias provide information on phenotypic diversity and disease mechanisms. Although cis-element polymorphisms and mutations are instructive, deciphering function remains challenging. Mutation of an intronic GATA motif (+9.5) in GATA2, encoding a master regulator of hematopoiesis, underlies an immunodeficiency associated with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Whereas an inversion relocalizes another GATA2 cis-element (-77) to the proto-oncogene EVI1, inducing EVI1 expression and AML, whether this reflects ectopic or physiological activity is unknown. We describe a mouse strain that decouples -77 function from proto-oncogene deregulation. The -77(-/-) mice exhibited a novel phenotypic constellation including late embryonic lethality and anemia. The -77 established a vital sector of the myeloid progenitor transcriptome, conferring multipotentiality. Unlike the +9.5(-/-) embryos, hematopoietic stem cell genesis was unaffected in -77(-/-) embryos. These results illustrate a paradigm in which cis-elements in a locus differentially control stem and progenitor cell transitions, and therefore the individual cis-element alterations cause unique and overlapping disease phenotypes.